Methods of inhibiting alu rna and therapeutic uses thereof

ABSTRACT

The presently-disclosed subject matter includes methods of identifying an Alu RNA inhibitor, and methods and compositions for inhibiting Alu RNA. Methods and compositions can be used for the treatment of geographic atrophy and other conditions of interest.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/701,450, now allowed, which is a 371 application of InternationalPatent Application No. PCT/US2011/038753, filed Jun. 1, 2011, whichclaims priority from U.S. Provisional Application Ser. No. 61/396,747,filed on Jun. 1, 2010; U.S. Provisional Application Ser. No. 61/432,110,filed Jan. 12, 2011; and U.S. Provisional Application Ser. No.61/432,948, filed Jan. 14, 2011. The entire disclosures of theseapplications are incorporated herein by this reference.

TECHNICAL FIELD

The presently-disclosed subject matter relates to uses of DICERoverexpression and the inhibition of Alu RNA.

INTRODUCTION

Geographic atrophy, an advanced form of age-related macular degenerationthat causes blindness in millions of people worldwide and for whichthere is no approved treatment, results from death of retinal pigmentedepithelium (RPE) cells. As described herein the present inventors showthat expression of DICER, an enzyme involved in microRNA (miRNA)biogenesis, is reduced in the RPE of human eyes with geographic atrophy,and that conditional ablation of Dicer1 induces RPE degeneration inmice. Surprisingly, ablation of seven other enzymes responsible formiRNA biogenesis or function does not induce such pathology. Instead,knockdown of DICER1 leads to accumulation of Alu repeat RNA in human RPEcells and of B1 and B2 (Alu-like elements) repeat RNAs in the RPE ofmice.

Alu RNA is dramatically increased in the RPE of human eyes withgeographic atrophy, and introduction of this pathological RNA inducesdeath of human RPE cells and RPE degeneration in mice.

Antisense oligonucleotides targeting Alu/B1/B2 RNAs inhibit DICER1depletion-induced RPE degeneration despite persistence of global miRNAdownregulation. DICER1 degrades Alu RNA, and Alu RNA loses the abilityto induce RPE degeneration in mice when digested by DICER1. Thesefindings reveal a novel miRNA-independent cell survival function forDICER1 via degradation of retrotransposon transcripts, introduce theconcept that Alu RNA can directly cause human pathology, and identifynew molecular targets for treating a major cause of blindness.

Age-related macular degeneration (AMD), which is as prevalent as cancerin industrialized countries, is a leading cause of blindness worldwide.In contrast to the neovascular form of AMD, for which many approvedtreatments exist¹, the far more common atrophic form of AMD remainspoorly understood and without effective clinical intervention².Extensive atrophy of the retinal pigment epithelium (RPE) leads tosevere vision loss and is termed geographic atrophy, the pathogenesis ofwhich is unclear. As described herein, the present inventors identifydysregulation of the RNase DICER1³ and the resulting accumulation oftranscripts of Alu elements, the most common small interspersedrepetitive elements in the human genome⁴, as a cause of geographicatrophy, and describe treatment strategies to inhibit this pathology invivo.

SUMMARY

The presently-disclosed subject matter meets some or all of the needsidentified herein, as will become evident to those of ordinary skill inthe art after a study of information provided in this document.

This Summary describes several embodiments of the presently-disclosedsubject matter, and in many cases lists variations and permutations ofthese embodiments. This Summary is merely exemplary of the numerous andvaried embodiments. Mention of one or more representative features of agiven embodiment is likewise exemplary. Such an embodiment can typicallyexist with or without the feature(s) mentioned; likewise, those featurescan be applied to other embodiments of the presently-disclosed subjectmatter, whether listed in this Summary or not. To avoid excessiverepetition, this Summary does not list or suggest all possiblecombinations of such features.

In some embodiments, the presently-disclosed subject matter includes amethod of identifying an Alu RNA inhibitor. The method can includeproviding a cell in culture wherein Alu RNA is upregulated; contactingthe cell with a candidate compound; and determining whether thecandidate compound results in a change in the Alu RNA. In someembodiments, the cell is an RPE cell. In some embodiments, the Alu RNAcan be upregulated by decreasing native levels of DICER polypeptides inthe cell. In some embodiments, the Alu RNA can be upregulated using heatshock stress. In some embodiments, the change in the Alu RNA is ameasurable decrease in Alu RNA, said change being an indication that thecandidate compound is an Alu RNA inhibitor.

In some embodiments, the presently-disclosed subject matter includes amethod of treating geographic atrophy, including inhibiting Alu RNAassociated with an RPE cell. In some embodiments, thepresently-disclosed subject matter includes a method of protecting anRPE cell, including inhibiting Alu RNA associated with the RPE cell. Insome embodiments, the RPE cell is of a subject having age-relatedmacular degeneration.

In some embodiments, the presently-disclosed subject matter includes amethod of treating a condition of interest, including inhibiting Alu RNAassociated with a cell of a subject. In some embodiments, the conditionof interest is selected from: geographic atrophy, dry age-relatedmacular degeneration, thallasemia, familial hypercholesterolemia, Dent'sdisease, acute intermittent porphyria, anterior pituitary aplasia, Apertsyndrome, Hemophilia A, Hemophilia B, glycerol kinase deficiency,autoimmune lymphoproliferative syndrome, X-linked agammaglobulinemia,X-linked severe combined immunodeficiency, adrenoleukodystrophy, Menkesdisease, hyper-immunoglobulin M syndrome, retinal blinding, Type 1anti-thrombin deficiency, Muckle-Wells syndrome, hypocalciurichypercalcemia and hyperparathyroidism, cholinesterase deficiency,hereditary desmoid disease, chronic hemolytic anemia, cystic fibrosis,branchio-oto-renal syndrome, lipoprotein lipase deficiency, CHARGEsyndrome, Walker Warburg syndrome, Complement deficiency, Mucolipidosistype II, Breast cancer, ovarian cancer, prostate cancer, von HippelLindau disease, Hereditary non-polyposis colorectal cancer, multipleendocrine neoplasia type 1, hereditary diffuse gastric cancer, hepatoma,neurofibromatosis type 1, acute myeloid leukemia, T-acute lymphoblasticleukemia, and Ewing sarcoma.

In some embodiments of the methods of the presently disclosed subjectmatter including inhibiting Alu RNA associated with a cell, theinhibiting Alu RNA comprises increasing levels of a DICER polypeptide inthe cell. In some embodiments, increasing levels of a DICER polypeptidecomprises overexpressing the DICER polypeptide in the cells. In someembodiments, increasing levels of a DICER polypeptide comprises using avector comprising a nucleotide encoding the DICER polypeptide. In someembodiments, the vector is a viral vector. In some embodiments, thevirus is selected from an adeno-associated virus, a lentivirus, and anadenovirus. In some embodiments, the vector is a plasmid vector. In someembodiments, the nucleotide encoding the DICER polypeptide is selectedfrom SEQ ID NO: 7 and SEQ ID NO: 8. In some embodiments, the DICERpolypeptide is selected from SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, 16,18, and 20. In some embodiments, the DICER polypeptide comprises afunctional fragment of the sequence of SEQ ID NO: 9, 18, or 20. In someembodiments, the DICER polypeptide comprises the following amino acidresidues of the polypeptide of SEQ ID NO: 9: 605-1922, 605-1912,1666-1922, 1666-1912, 605-1786 and 1800-1922, 605-1786 and 1800-1912,1666-1786 and 1800-1922, 1666-1786 and 1800-1912, 1276-1922, 1276-1912,1276-1786 and 1800-1922, 1276-1786, 800-1912, 1275-1824, or 1276-1824.

In some embodiments of the methods of the presently disclosed subjectmatter including inhibiting Alu RNA associated with a cell, theinhibiting Alu RNA comprises increasing levels of a DICER polypeptidecomprises using DICER mRNA or a functional fragment thereof. In someembodiments, the DICER mRNA has the sequence of SEQ ID NO: 17, 19, or21. In some embodiments, the DICER mRNA encodes a DICER polypeptide, forexample, the DICER polypeptide of SEQ ID NO: 9, 18, or 20, or afunctional fragment thereof.

In some embodiments of the methods of the presently disclosed subjectmatter including inhibiting Alu RNA associated with a cell, theinhibiting Alu RNA comprises administering an oligonucleotide targetingAlu RNA. In some embodiments, the oligonucleotide has a sequenceincluding a sequence selected from SEQ ID NO: 22, 23, 24, 25, and 26. Insome embodiments, at least two oligonucleotides are administered. Thepresently-disclosed subject matter further includes an isolatedoligonucleotide that inhibits the expression of Alu RNA, including asequence selected from SEQ ID NO: 22, 23, 24, 25, and 26 and includingabout 29 to 100 nucleotides.

In some embodiments of the methods of the presently disclosed subjectmatter including inhibiting Alu RNA associated with a cell, theinhibiting Alu RNA comprises administering an siRNA targeting Alu RNA.In some embodiments, the siRNA includes a first strand having a sequenceselected from SEQ ID NO: 1, 2, 3, 4, 5, and 6. The presently-disclosedsubject matter further includes an isolated double-stranded RNA moleculethat inhibits expression of Alu RNA, wherein a first strand of thedouble-stranded RNA comprises a sequence selected from SEQ ID NO: 1, 2,3, 4, 5, and 6 and including about 19 to 25 nucleotides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 DICER1 deficit in geographic atrophy induces RPE degeneration. a,DICER1 mRNA abundance, relative to 18S rRNA, monitored by real-timeRT-PCR, was lower in the retinal pigmented epithelium (RPE) of humaneyes with geographic atrophy (GA; n=10) compared to the RPE of normalhuman eyes without GA (n=11). P=0.004 by Mann Whitney U test. Theabundance of DROSHA, DGCR8, and EIF2C2 (encoding AGO2) mRNA transcriptsin the RPE was not significantly different (P>0.11 by Mann Whitney Utest) in human eyes with geographic atrophy and control eyes. Transcriptabundance quantified by real-time RT-PCR and normalized to 18S rRNA andto control eye levels. n=10-11. b, Relative quantification of DICER1protein abundance, relative to Vinculin, assessed by Western blotting(Supplementary FIG. 1), was lower in the RPE of human eyes withgeographic atrophy (GA; n=4) compared to the RPE of normal human eyeswithout GA (n=4). P=0.003 by Student t test. c, Immunohistochemistry forDICER1 (blue) showed reduced protein abundance in the RPE of human eyeswith GA compared to normal eyes without GA. d, Fundus photographs showextensive RPE degeneration in BEST1 Cre; Dicer1^(f/f) mice but not inlittermate control mice. e, Toluidine blue-stained sections show markedRPE degeneration in BEST1 Cre; Dicer1^(f/f) mice compared to normal RPEarchitecture in control mice. Arrowheads point to basal surface of RPE.f, Flat mounts of the RPE and choroid stained with antibodies againstzonula occludens-1 (ZO-1; red) show marked disruption of the RPEmonolayer architecture in BEST1 Cre; Dicer^(f/f) mice compared to theuniformly tesselated RPE layer in littermate control mice. g, Fundusphotographs show RPE degeneration in Dicer1^(f/f) mice followingsubretinal injection of AAV1-BEST1-Cre but not AAV1-BEST1-GFP. h,Toluidine blue-stained sections show marked degeneration of RPE andphotoreceptor outer segments in Dicer1^(f/f) mice following subretinalinjection of AAV1-BEST1-Cre but not AAV1-BEST1-GFP. i, Flat mounts showmarked increase in RPE cell size and distortion of RPE cell shape inDicer1^(f/f) mice following subretinal injection of AAV1-BEST1-Cre butnot AAV1-BEST1-GFP. RPE cell borders outlined by ZO-1 staining (red).Nuclei stained blue with Hoechst 33342. Representative images shown.n=16-32 (d-f); 10-12 (g-i). Scale bars, (c,e,h), 10 μm; (f,i) 20 μm. j,Transfection of adenoviral vector coding for Cre recombinase (Ad-Cre) inRPE cells isolated from Dicer1^(f/f) mice resulted in loss of cellviability, as monitored by MTS assay at 7 days, compared to transfectionwith Ad-Null or untreated (no Tx) cells. k, Transfection of antisenseoligonucleotide (as) targeting DICER1 into human RPE cells resulted inincreasing loss of cell viability over time compared to scrambledsequence antisense (Ctrl as)-treated cells. n=6-8.

FIG. 2 Alu RNA accumulation in geographic atrophy triggered by DICERreduction. a, Immunohistochemistry with anti-double stranded RNA (dsRNA)antibody (J2) shows abundant accumulation of dsRNA (blue staining) inthe retinal pigmented epithelium (RPE) of a human eye with geographicatrophy. b, Lack of immunolabeling with an isotype antibody in the sameeye with geographic atrophy confirms specificity of dsRNA staining c,d,dsRNA is immunolocalized (blue staining) in the RPE and sub-RPE deposits(drusen) of a human eye with geographic atrophy (c) but not in the RPEof a normal (control) eye (d). Scale bars, (a-d), 10 μm. n=10 (a-d) e,PCR amplification of dsRNA immunoprecipitated by J2 antibody from RPEisolates from human eyes with geographic atrophy and normal eyes yieldedamplicons with sequence homology to Alu sequences (Supplementary FIG.S7) in eyes with geographic atrophy but not in normal eyes. Waternegative control (−) showed no amplification and positive control (+)recombinant dsRNA showed predicted amplicon. f, Alu RNA abundance,relative to 18S rRNA, monitored by real-time RT-PCR, was higher in theRPE of human eyes with geographic atrophy compared to the RPE of normalhuman eyes without GA (n=7). P<0.05 by Student t test. There was nosignificant difference in Alu RNA abundance in the neural retina ofthese two patient groups. Values normalized to relative abundance innormal eyes.

FIG. 3 DICER1 degrades Alu RNA. a, Transfection of antisenseoligonucleotide (as) targeting DICER1 into human RPE cells induced atime-dependent increase in the abundance of Alu RNA transcripts. b, c,Transfection of adenoviral vector coding for Cre recombinase (Ad-Cre)into mouse RPE cells isolated from Dicer1^(f/f) mice increased, in thenucleus (b) and the cytoplasm (c), the abundance of B1 and B2 RNAs, theAlu-like repetitive elements in the mouse, compared to cells transfectedwith adenoviral vector coding for green fluorescent protein (Ad-GFP). d,DICER1 as treatment of human RPE cells upregulated Alu RNA levels inboth the nucleus (Nuc) and cytoplasm (Cyt). e, Alu RNA isolated andcloned from the RPE of human eye with geographic atrophy was degraded byrecombinant DICER1 digestion (+) as visualized by agarose gelelectrophoresis. Digestion with heat denatured DICER1 did not degradeAlu RNA. Image representative of 6 experiments. f, The increasedabundance of Alu RNA in human RPE cells transfected with plasmid codingfor Alu (pAlu) compared to pNull or no treatment (no Tx) at 24 h wasreduced by co-transfection with pDICER1. *P<0.05. n=4-8 (a-d, f). RNAabundance was quantified by real-time RT-PCR, normalized to 18S rRNAlevels, and normalized to levels in control as-treated (for Alu) orAd-GFP-infected cells (for B elements).

FIG. 4 DICER1 protects RPE cells from Alu RNA cytotoxicity. a,Transfection of mouse or human retinal pigmented epithelium cells (mRPEor hRPE) with plasmid coding for Alu RNA (pAlu) compromised cellviability. b, Subretinal administration of pAlu induced RPE degenerationin wild-type mice whereas pNull did not do so. Fundus photograph (toprow) shows area of degeneration in pAlu injected eye compared to thenormal appearance in pNull. Flat mount preparations stained withanti-zonula occludens-1 antibody (ZO-1, red, bottom row) show markeddistortion of RPE cell shape and size compared to pNull-injected eye. c,Alu RNA induced dose-dependent increase in cell death of human RPEcells. d, Cell death of human RPE cells induced by transfection of pAluwas inhibited by co-transfection with pDICER1 but not pNull. (a,c,d)Cell viability monitored by MTS assay at 2 days. Values normalized tonull plasmid (pNull) transfected or vehicle treated cells. *P<0.05 byStudent t test. n=4-6. e, Subretinal co-administration of pDICER1, butnot of pNull, inhibited pAlu induced RPE degeneration in wild-type mice.f, Subretinal administration of Alu RNA isolated and cloned from the RPEof a human eye with geographic atrophy (GA) induced RPE degeneration inwild-type mice whereas subretinal injection of vehicle did not. g,Subretinal injection of this Alu RNA, when subjected to cleavage byDICER1, did not induce RPE degeneration in wild-type mice whereas AluRNA subjected to mock cleavage by DICER1 did do so, as evident on fundusphotography (top row) or flat mount preparation (bottom row). Area ofdegeneration outlined by blue arrowheads in fundus photographs (b, e-g).Scale bars (20 μm). n=10-15 (b, e-g).

FIG. 5 DICER1 dyregulation induces RPE cell death via Alu RNAaccumulation. a, Loss of human RPE cell viability, as monitored by MTSassay, induced by transfection of antisense oligonucleotide (as)targeting DICER1 was rescued by co-transfection of Alu RNA as. Levelsnormalized or compared to transfection with control (Ctrl) antisenseoligonucleotide. b, Alu RNA as inhibited accumulation of Alu RNA inducedby DICER1 as. c, Ad-Cre but not Ad-Null induced loss of cell viabilityof Dicer1^(f/f) mouse RPE cells. This was rescued by transfection ofantisense oligonucleotide targeting B1 and B2 RNAs but not by control(Ctrl) antisense oligonucleotide. Levels normalized to untreated cells(no Tx). d, B1/B2 RNA as inhibited accumulation of B1 and B2 RNAsinduced by Ad-Cre-induced Dicer1 depletion. *P<0.05 by Student t test.n=4-6 (a-d). d, Subretinal AAV-BEST1-Cre administration induced RPEdegeneration (blue arrowheads in fundus photograph on top row and markedincrease in RPE cell size and distortion of RPE cell shape in ZO-1stained (red) RPE flat mounts (bottom row) in Dicer1^(f/f) mice 20 daysafter injection. Subretinal administration of cholesterol-conjugatedB1/B2 as, but not Ctrl as, 10 days after AAV-BEST1-Cre injectioninhibited RPE degeneration (e) and abundance of B1/B2 RNAs in the RPE ofthese mice, as monitored by real-time RT-PCR at 10 days after asinjection, normalized to 18S rRNA levels, and normalized to levels ineyes treated with cholesterol-conjugated Ctrl as (f). n=8 (e,f). Scalebar, 20 μm. (e). g, DICER1 as treatment of human RPE cells led to globalreduction of miRNA expression at 2 days compared to Ctrl as. There wasno significant difference in miRNA abundance between Alu as and Ctrlas-treated DICER1 depleted cells. n=3.

FIG. 6 DICER1 levels in RPE are reduced in geographic atrophy. Westernblots of macular RPE lysates from individual human donor eyes show thatDICER1 protein abundance, normalized to the levels of the housekeepingprotein Vinculin, are reduced in geographic atrophy (GA) compared toage-similar control human eyes without age-related macular degeneration.

FIG. 7 DICER1 levels in neural retina are unchanged in geographicatrophy. a, DICER1 mRNA abundance in the neural retina, as monitored byreal-time RT-PCR, was not significantly different (P>0.05 by MannWhitney U test) between normal human retinas and those with geographicatrophy. Levels normalized to 18S rRNA abundance and to normal retinas.n=7. b-e, DICER1 protein immunolocalization in the neural retina was notdifferent between human eyes with geographic atrophy (b) and normal (d)eyes. Specificity of DICER1 staining was confirmed by absence ofreaction production with isotype control antibody (c,e). Representativeimages shown. n=8. Scale bars (20 μm, b-e).

FIG. 8 DICER1 is not generically downregulated in retinal diseases.Immunolocalization studies revealed abundant DICER1 protein expression(blue, left column) in the RPE in the eye of an 85-year-old man withBest disease (vitelliform macular dystrophy), a 68-year-old man withretinal detachment secondary to choroidal melanoma, and a 72-year-oldwoman with retinitis pigmentosa. Specificity of DICER1 staining wasconfirmed by absence of reaction production with isotype controlantibody (right column). Representative images shown. n=13. Scale bars(10 μm). Dicer1 mRNA expression in the RPE was not significantly (NS)different in Ccl2^(−/−) Ccr2^(−/−) mice or Cp^(−/−) Heph^(−/−) micecompared to their background strains. Transcript abundance quantified byreal-time RT-PCR and normalized to 18S rRNA and to control eye levels.n=6. NS, not significant.

FIG. 9 Cre recombinase expression does not induce retinal pigmentedepithelium (RPE) degeneration. Subretinal administration ofadeno-associated viral vector coding for Cre recombinase directed by theBEST1 promoter (AAV1-BEST1-Cre) in wild-type mice did not induce retinaltoxicity that was evident on fundus photography (top left) and did notdisrupt the tiling pattern of the RPE monolayer (top right). Circularflash artifact is seen in the centre of the fundus photograph. RPE cellborders delineated by staining with anti-ZO-1 antibody (red) and nucleistained by Hoechst 33342 (blue). RPE flat mounts show successful Crerecombinase expression (red) following subretinal injection ofAAV1-BEST1-Cre in wild-type (bottom left) and Dicer1^(f/f) (bottomright) mouse eyes. Representative images shown. n=8-10. Scale bar (20μm).

FIG. 10 Retinal pigmented epithelium (RPE) cell dysmorphology in humanage-related macular degeneration eye with atrophy. In contrast to thewell tessellated RPE cell monolayer observed in a normal human eye(right), marked changes in RPE cell size and shape are observed in thehuman eye with geographic atrophy (left). These changes resemble thoseobserved in eyes of mice wherein Dicer1 has been depleted in the RPE.RPE cell borders delineated by staining with anti-ZO-1 antibody (green)and nuclei stained by propidium iodide (red). Representative imageshown. n=8. Scale bar, 50 μm.

FIG. 11 Conditional ablation of Drosha, Dgcr8, or Ago2 in the retinalpigmented epithelium (RPE) does not induce degeneration as seen inDicer1-ablated mice. Fundus photographs (left column) show nosignificant degeneration following subretinal injection of AAV-BEST1-Crein mice “foxed” for Drosha, DGCR8, or Ago2. Circular flash artifacts areseen near the centre of the fundus photographs. Injection site woundappears white in the fundus photograph of the Ago2^(f/f) eye. RPE flatmounts (middle column) stained with anti-ZO-1 antibody (red) and Hoechst33342 (blue) show normal tiling pattern of RPE with no gross disturbanceof cell size or shape. RPE flat mounts (right column) stained withanti-Cre recombinase antibody (red) and Hoechst 33342 (blue) showssuccessful Cre expression in these mice eyes. Representative imagesshown. n=8-12. Scale bar (20 μm).

FIG. 12 Deficiency of Ago1, Ago3, Ago4, or Tarbp2 does not induce RPEdegeneration. Mice deficient in Ago1 Ago3 Ago4, or Tarbp2 have normalretinal appearance on fundus photography (top row) and normal RPEmonolayer architecture on ZO-1 stained (red) flat mounts (bottom row).Circular flash artifact is seen in the centre of the fundus photographs.Scale bar, 20 μm.

FIG. 13 DICER1 mutant cells impaired in miRNA biogenesis do not havecompromised cell viability. There was no difference in baseline cellviability between HCT-DICER1^(ex5) cells, which are impaired in miRNAbiogenesis¹, and parent HCT116 cells over 3 days of analysis of cellproliferation. n=3. NS, not significant.

FIG. 14 Human geographic atrophy eye retinal pigmented epithelia containAlu RNA sequences. a, Top: Typical Alu element with conserved structuralregions (adapted from ref. 2). The left arm consists of RNA polymeraseIII binding sites (Box A and Box B). The right arm occasionally containsa terminal poly A tail that may be interspersed with non-A bases. The 5′and 3′ regions of the Alu element are linked by a mid-stretch A-richsequence. Bottom: Representative Alu cDNA (Sequence 1). The conservedregions mentioned above are highlighted and correspond to the colouredboxes in the top figure. b, Alignment of Alu cDNA Sequences 1 and 2isolated from human eyes with geographic atrophy to Alu Sq consensussequence. These sequences contain the highly conserved 5′ Alu consensuselements (5′ characteristic Alu region—blue; RNA polymerase III promoterB box—red), with extensive heterogeneity located 3′ to the mid-sequencepoly-A stretch that have been reported to exist in Alu sequences^(3,4).

FIG. 15 J2 anti-dsRNA antibody recognizes Alu RNA. a, Alu RNA duplexisolated and cloned from the retinal pigmented epithelium (RPE) of ahuman eye with geographic atrophy was recognized by J2 anti-dsRNAantibody in an immuno-dot blot format. J2 antibody did not recognizerRNA or tRNA (negative controls), but did recognize RNA duplexes of325-bp or 1-kbp in length (positive controls). b, Immunofluorescentimaging of human RPE cells transfected with pAlu shows that J2recognizes Alu expressed in these cells (left panel). Specificity ofstaining confirmed by absence of staining with isotype control antibody(middle panel) and by the absence of immunodetection followingtransfection with pNull (right panel). Representative images shown. n=3.Scale bar (20 μm).

FIG. 16 Confirmation of lack of DNA contamination in Alu RNA PCR. Therelative abundance of Alu RNA in the RPE of human eyes with humangeographic eyes was presented in FIG. 2 f. Shown above is the detectionof the PCR product band for a sample of human geographic atrophy RPEthat underwent reverse transcription (RT+). No amplification wasdetected in the negative controls where reverse transcriptase (RT−) wasomitted or where water alone was analyzed. These data demonstrate theabsence of DNA contamination in the sample.

FIG. 17 Validation of DICER1 knockdown. Transfection of DICER1 antisenseoligonucleotides (as) into human RPE cells knocks down DICER1 proteinabundance, as monitored by Western blot analysis, over 2 days.Efficiency of protein loading is monitored by blotting for thehousekeeping Vinculin protein. Representative of 3 experiments.

FIG. 18 DICER1 is expressed in nucleus and cytoplasm. a, Western blotshows expression of DICER1 in both the nuclear and cytoplasmic fractionsof human RPE cells. Blotting of the same protein sample reveals thepresence of Tubulin in the cytoplasmic fraction and not in the nuclearfraction. b, Merged images (bottom row) of DICER1 immunofluorescence(red, top row) and nuclear DAPI fluorescence (middle row) confirmexpression of DICER1 in both the nucleus and the cytoplasm of human RPEcells. Representative images shown. Scale bar, 10 μm.

FIG. 19 Retrotransposons and repetitive RNAs are not genericallyactivated in geographic atrophy or by DICER1 depletion. In the RPE ofhuman eyes with geographic atrophy (GA, n=7), there was no significantincrease in the abundance of RNAs coded by LINE L1.3, a longinterspersed repetitive element, human endogenous retrovirus-W envelope(HERV-WE1), a long terminal repeat retrotransposon, or hY3, a repetitivesmall cytoplasmic Ro RNA compared to normal human eyes (top, n=8). TheseRNAs also were not upregulated by DICER1 antisense (as) knockdown,compared to control (Ctrl) as treatment, in human RPE cells (bottom).n=3. Transcript abundance monitored by real-time RT-PCR and normalizedto 18S rRNA levels.

FIG. 20 Alu RNA induced by DICER1 depletion is RNA Pol III derived. a,The upregulation of Alu RNA in RPE cells treated with antisense (as)oligonucleotides targeting DICER1, compared to control (Ctrl), isinhibited by treatment with the Pol III inhibitor tagetitoxin (tagetin),but not by the Pol II inhibitor α-amanitin. *, P<0.05, NS, notsignificant, compared to treatment with DICER1 as treatment alone. b,Northern blot (NB) shows that the abundance of Alu RNA species in theRPE of a human eye with geographic atrophy (GA) is greater than innormal human eye RPE, and is principally approximately 300 nucleotideslong, consistent with the length of a non-embedded Pol III derivedtranscript. Reprobing these samples with a probe corresponding to the “Sregion” of the 7SL RNA gene that is not present in Alu elements showsthat 7SL RNA abundance is not different between the RPE of normal and GAhuman eyes. Abundance of U6 RNA in GA and normal eyes shows loadingefficiency. c, Northern blot shows that Alu probe detects in vitrotranscribed Alu RNA but not 7SL RNA in mouse liver (which lacksprimate-specific Alu), and reprobing these samples confirms specificityof the 7SL probe. d, DICER1 knockdown by antisense (as) oligonucleotidesin human RPE cells does not, compared to control (Ctrl) as treatment,induce upregulation of several Pol II-transcribed genes (ADAR2, NICN,NLRP, SLFN 11) that contain embedded Alu sequences in their exons. n=3.

FIG. 21 7SL RNA is not regulated in geographic atrophy or by inhibitionof DICER1 or Alu. a, 7SL RNA abundance was not different in the RPE ofhuman eyes with geographic atrophy (GA) compared to the RPE of normalhuman eyes without GA (n=8). b, 7SL RNA abundance was not different inhuman RPE cells transfected with antisense oligonucleotide (as)targeting DICER1 from those transfected with control (Ctrl) as. n=3. c,7SL RNA abundance was not different in human RPE cells transfected withantisense oligonucleotide (as) targeting Alu from those transfected withcontrol (Ctrl) as. n=3. 7SL RNA abundance, relative to 18S rRNA, wasmonitored by real-time RT-PCR. NS, not significant by Student t test.

FIG. 22 Overexpression of B1 or B2 RNA induces RPE degeneration.Subretinal transfection of pB1 or pB2 RNAs, but not of pNull, inducesRPE degeneration in wild-type mice. Top row shows fundus photographsdemonstrating areas of degeneration outlined by blue arrowheads. Bottomrow shows ZO-1 stained (red) RPE flat mounts demonstrated markeddegeneration and disarray of the RPE cells in mice overexpressing B1 orB2 RNAs. Circular flash artifact is seen in the centre of the fundusphotographs. n=4. Representative images shown. Scale bar, 20 μm.

FIG. 23 Alu RNA enters retinal pigmented epithelium (RPE) cells in vivo.Subretinal administration of Alu RNA in wild-type mice achieved RPE celldelivery at 8 h after injection as monitored by real-time RT-PCR inisolated cell lysates (n=3).

FIG. 24 Human GA Alu dsRNA does not induce RPE degeneration when cleavedby DICER1. a, Subretinal administration of a fully complementarysynthetic Alu RNA (dsRNA) corresponding to the sequence of an Alu RNAisolated from a human eye with geographic atrophy (GA) induces RPEdegeneration in wild-type mice. Vehicle administration does not damagethe retina. Top panels show fundus photographs with the area of RPEdegeneration outlined by blue arrowheads. Circular flash artifact isseen in the centre of the fundus photographs. Bottom panels show ZO-1stained (red) RPE flat mounts that are well arrayed in vehicle (bottom)but disorganized in Alu dsRNA (top). b, This Alu dsRNA did not induceRPE degeneration when it was first subjected to cleavage by recombinantDICER1. However, when subjected to mock cleavage by DICER1, this AludsRNA did induce RPE degeneration. n=4. Representative images shown.Scale bar, 20 μm.

FIG. 25 RPE degeneration does not occur in response to a variety ofstructured RNAs. Subretinal transfection of transfer RNA (tRNA) or ofplasmids coding for 7SL RNA, pri-miRNA-29b1 or pri-miRNA26a2 inwild-type mice did not induce retinal toxicity that was evident onfundus photography. Circular flash artifact is seen in the centre of thefundus photographs. N=4. Representative images shown.

FIG. 26 Alu RNA does not cause RPE degeneration via TLR3. a, Westernblot shows that transfection of pAlu or pNull does not induce TLR3phosphorylation, relative to the levels of the housekeeping proteinVinculin, in human RPE cells. b, Subretinal transfection of pAlu inducedRPE degeneration in Tlr3−/− mice where pNull transfection did not do so.Representative images shown. n=4. Scale bar, 20 μm.

FIG. 27 DICER1 reduction or Alu RNA augmentation induces caspase-3activation. a, Immunolocalization of activated caspase-3 (red) in theRPE of human eyes with geographic atrophy (left panel). Specificity ofimmunolabeling revealed by absence of staining with isotype controlantibody (middle panel) and in control eyes stained with antibodyagainst cleaved caspase-3 (right panel). Autofluorescence of RPE andchoroid seen in green channel. Nuclei stained by DAPI (blue). b, Flatmounts of BEST1 Cre; Dicer1^(f/f) mice show evidence of caspase-3activation (red staining, top left panel). Specificity of immunolabelingrevealed by absence of staining with isotype control antibody (top rightpanel). No caspase-3 activation was detectable in the RPE of littermatecontrol BEST1 Cre or Dicer1^(f/f) mice (bottom panels). c, Human RPEcells transfected with pAlu showed evidence of caspase-3 activation (redstaining, top left panel). DAPI (blue staining) and merged images arealso shown. Scale bars (20 μm, a,b; 10 μm, c). Representative imagesshown. n=4-6. d, Exposure of human RPE cells to Alu RNA induceddose-dependent increase in caspase-3 activation, as monitored byfluorometric plate assay. n=3, *P<0.05 compared to vehicle by Student ttest. e, Transfection of human RPE cells with pAlu induced increase incaspase-3 activation. n=3, *P=0.47 by Student t test.

FIG. 28 Alu RNA cleavage fragments do not modulate RPE degeneration. a,Transfection of pAlu induced cell death in human RPE cells.Cotransfection of DICER1-cleaved Alu RNA fragments did not change thedegree of cell death. n=3. b, Subretinal transfection of DICER1-cleavedAlu RNA fragments (Frag) in wild-type mice did not cause RPEdegeneration as seen by fundus photography (top left) or ZO-1-stained(red) RPE flat mounts (bottom left). Cotransfections of these fragmentsdid not prevent the RPE degeneration induced by pAlu in wild-type mice(right panels). n=4. Representative images shown. Scale bar, 20 μm.

FIG. 29 Impaired DICER1 processing of microRNAs does not increase AluRNA abundance or modulate Alu RNA cytotoxicity. a, There was nosignificant difference (P>0.05) in Alu RNA transcript abundance betweenHCT116 parent cells and HCT mutant cells carrying a mutation in exon 5(ex5) of DICER1 which renders it incapable of processing microRNAs. b,Transfection of anti-sense oligonucleotide (as) targeting DICER1 intoHCT116 cells increased the abundance of Alu RNA transcripts compared tocontrol anti-sense oligonucleotide (Ctrl as) at 48 h. Transcriptabundance monitored by real-time RT-PCR and normalized to 18S rRNAlevels. c, Alu RNA induced similar levels of cell death in HCT116 parentand HCT-DICER1^(ex5) cells. *P<0.05 by Student t test. n=4-6.

FIG. 30 Oxidative stress downregulates DICER1 in human RPE cells. Humanretinal pigmented epithelium (RPE) cells exposed to varyingconcentrations of hydrogen peroxide (H₂O₂) display a dose- andtime-dependent reduction in DICER1 mRNA abundance, as monitored byreal-time RT-PCR and normalized to 18S rRNA levels. n=3.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 is an embodiment of a first strand of an siRNA provided inaccordance with the presently-disclosed subject matter.

SEQ ID NO: 2 is an embodiment of a first strand of an siRNA provided inaccordance with the presently-disclosed subject matter.

SEQ ID NO: 3 is an embodiment of a first strand of an siRNA provided inaccordance with the presently-disclosed subject matter.

SEQ ID NO: 4 is an embodiment of a first strand of an siRNA provided inaccordance with the presently-disclosed subject matter.

SEQ ID NO: 5 is an embodiment of a first strand of an siRNA provided inaccordance with the presently-disclosed subject matter.

SEQ ID NO: 6 is an embodiment of a first strand of an siRNA provided inaccordance with the presently-disclosed subject matter.

SEQ ID NO: 7 is nucleotide sequence encoding a human DICER polypeptide,including all untranslated regions (GenBank Accession NumberNM_(—)177438).

SEQ ID NO: 8 is a cDNA sequence encoding a human DICER polypeptide.

SEQ ID NO: 9 is a polypeptide sequence for a human DICER polypeptide.

SEQ ID NO: 10 is a polypeptide sequence for a human DICER polypeptide,including residues 1276-1922 of SEQ ID NO: 9.

SEQ ID NO: 11 is a polypeptide sequence for a human DICER polypeptide,including residues 605-1922 of SEQ ID NO: 9.

SEQ ID NO: 12 is a polypeptide sequence for a human DICER polypeptide,including residues 1666-1922 of SEQ ID NO: 9.

SEQ ID NO: 13 is a polypeptide sequence for a human DICER polypeptide,including residues 1666-1912 of SEQ ID NO: 9.

SEQ ID NO: 14 is a polypeptide sequence for a human DICER polypeptide,including residues 1666-1786 and 1800-1912 of SEQ ID NO: 9.

SEQ ID NO: 15 is a polypeptide sequence for a human DICER polypeptide,including residues 1275-1824 of SEQ ID NO: 9.

SEQ ID NO: 16 is a polypeptide sequence for a human DICER polypeptide,including residues 1276-1824 of SEQ ID NO: 9.

SEQ ID NO: 17 is an mRNA sequence encoding a human DICER polypeptide.

SEQ ID NO: 18 is a polypeptide sequence for a Schizosaccharomyces pombeDICER polypeptide.

SEQ ID NO: 19 is an mRNA sequence encoding a Schizosaccharomyces pombeDICER polypeptide.

SEQ ID NO: 20 is a polypeptide sequence for a Giardia lamblia DICERpolypeptide.

SEQ ID NO: 21 is an mRNA sequence encoding a Giardia lamblia DICERpolypeptide.

SEQ ID NO: 22 is an embodiment of an antisense oligonucleotide sequenceprovided in accordance with the presently-disclosed subject matter.

SEQ ID NO: 23 is an embodiment of an antisense oligonucleotide sequenceprovided in accordance with the presently-disclosed subject matter.

SEQ ID NO: 24 is an embodiment of an antisense oligonucleotide sequenceprovided in accordance with the presently-disclosed subject matter.

SEQ ID NO: 25 is an embodiment of an antisense oligonucleotide sequenceprovided in accordance with the presently-disclosed subject matter.

SEQ ID NO: 26 is an embodiment of an antisense oligonucleotide sequenceprovided in accordance with the presently-disclosed subject matter.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

The presently-disclosed subject matter includes methods for identifyingAlu RNA inhibitors, and methods and compositions for inhibiting Alu RNAand therapeutic uses thereof

As disclosed herein, Alu RNA (including Alu repeat RNA in human cellsand B1 and B2, Alu-like element repeat RNAs) increases are associatedwith cells that are associated with certain conditions of interest. Forexample, Alu RNA increase is associated with the retinal pigmentepithelium (RPE) cells of eyes with geographic atrophy. This increase ofAlu RNA induces the death of RPE cells. Methods and compositionsdisclosed herein can protect a cell from Alu RNA-triggered cell death,thereby treating conditions associated with such cell death.

The presently-disclosed subject matter further includes methods usefulfor identifying an Alu RNA inhibitor and uses of such inhibitors,including therapeutic and protective uses. In some embodiments, themethod makes use of a cultured cell wherein Alu RNA is upregulated.Candidate compounds can be screened using the cultured cell to determineefficacy as antagonists of Alu RNA. Candidate compounds include, forexample, small molecules, biologics, and combinations thereof, such ascompositions including multiple compounds. The term small molecules isinclusive of traditional pharmaceutical compounds. The term biologics isinclusive of polypeptides and nucleotides.

In some embodiments, the screening method includes providing a cell inculture wherein Alu RNA is upregulated; and contacting a candidatecompound with the cell. The method can further include identifying achange in Alu RNA. For example, a measurable change in Alu RNA levelscan be indicative of efficacy associated with the candidate compound. Insome embodiments, wherein the change in the Alu RNA is a measurabledecrease in Alu RNA, the change is an indication that the candidatecompound is an Alu RNA inhibitor. Such Alu RNA inhibitors can haveutility for therapeutic applications as disclosed herein.

In some embodiments, the Alu RNA can be upregulated by decreasing nativelevels of DICER polypeptides in the cell using methods known to thoseskilled in the art. In some embodiments, the Alu RNA associated withcultured cell can be upregulated by using heat shock stress usingmethods known to those skilled in the art. In some embodiments, thecultured cell is an RPE cell.

Methods and compositions of the presently-disclosed subject matter fortreating a condition of interest include inhibiting Alu RNA associatedwith a cell, such as a cell of a subject in need of treatment. Examplesof conditions of interest include, but are not limited to: geographicatrophy, dry age-related macular degeneration, thallasemia, familialhypercholesterolemia, Dent's disease, acute intermittent porphyria,anterior pituitary aplasia, Apert syndrome, Hemophilia A, Hemophilia B,glycerol kinase deficiency, autoimmune lymphoproliferative syndrome,X-linked agammaglobulinemia, X-linked severe combined immunodeficiency,adrenoleukodystrophy, Menkes disease, hyper-immunoglobulin M syndrome,retinal blinding, Type 1 anti-thrombin deficiency, Muckle-Wellssyndrome, hypocalciuric hypercalcemia and hyperparathyroidism,cholinesterase deficiency, hereditary desmoid disease, chronic hemolyticanemia, cystic fibrosis, branchio-oto-renal syndrome, lipoprotein lipasedeficiency, CHARGE syndrome, Walker Warburg syndrome, Complementdeficiency, Mucolipidosis type II, Breast cancer, ovarian cancer,prostate cancer, von Hippel Lindau disease, Hereditary non-polyposiscolorectal cancer, multiple endocrine neoplasia type 1, hereditarydiffuse gastric cancer, hepatoma, neurofibromatosis type 1, acutemyeloid leukemia, T-acute lymphoblastic leukemia, and Ewing sarcoma.

As used herein, the terms treatment or treating relate to any treatmentof a condition of interest, including but not limited to prophylactictreatment and therapeutic treatment. As such, the terms treatment ortreating include, but are not limited to: preventing a condition ofinterest or the development of a condition of interest; inhibiting theprogression of a condition of interest; arresting or preventing thedevelopment of a condition of interest; reducing the severity of acondition of interest; ameliorating or relieving symptoms associatedwith a condition of interest; and causing a regression of the conditionof interest or one or more of the symptoms associated with the conditionof interest.

As used herein, the term “subject” refers to a target of treatment. Thesubject of the herein disclosed methods can be a vertebrate, such as amammal, a fish, a bird, a reptile, or an amphibian. Thus, the subject ofthe herein disclosed methods can be a human or non human. Thus,veterinary therapeutic uses are provided in accordance with thepresently disclosed subject matter.

In some embodiments, the condition of interest is geographic atrophy andthe cell is an RPE cell. In this regard, a subject having age-relatedmacular degeneration can be treated using methods and compositions asdisclosed herein.

As will be understood by those skilled in the art upon studying thisapplication, inhibition of Alu RNA associated a cell can be achieved ina number of manners. For example, in some embodiments, inhibiting AluRNA associated with a cell comprises increasing levels of a DICERpolypeptide in the cell, for example, by overexpressing the DICERpolypeptide in the cell. For another example, a DICER mRNA could beused. For another example, in some embodiments, inhibiting Alu RNAassociated with a cell comprises administering an oligonucleotide or asmall RNA molecule targeting the Alu RNA. As used herein, inhibiting AluRNA associated with a cell refers to a reduction in the levels of AluRNA inside and/or outside the cell in the extracellular space.

The term DICER Polypeptide refers to polypeptides known to those ofordinary skill in the art as DICER, including, but not limited topolypeptides comprising the sequences of SEQ ID NO: 9, 18, and 20, andfunctional fragments or functional variants thereof.

It is noted that one of ordinary skill in the art will be able toreadily obtain publicly-available information related to DICER,including relevant nucleotide and polypeptide sequences included inpublicly-accessible databases, such as GENBANK®. Some of the sequencesdisclosed herein are cross-referenced to GENBANK® accession numbers,e.g., GenBank Accession Number NM_(—)177438. The sequencescross-referenced in the GENBANK® database are expressly incorporated byreference as are equivalent and related sequences present in GENBANK® orother public databases. Also expressly incorporated herein by referenceare all annotations present in the GENBANK® database associated with thesequences disclosed herein. Unless otherwise indicated or apparent, thereferences to the GENBANK® database are references to the most recentversion of the database as of the filing date of this application.

The terms “polypeptide”, “protein”, and “peptide”, which are usedinterchangeably herein, refer to a polymer of the 20 protein aminoacids, or amino acid analogs, regardless of its size. The terms“polypeptide fragment” or “fragment”, when used in reference to areference polypeptide, refers to a polypeptide in which amino acidresidues are deleted as compared to the reference polypeptide itself,but where the remaining amino acid sequence is usually identical to thecorresponding positions in the reference polypeptide. Such deletions canoccur at the amino-terminus (e.g., removing residues 1-604, 1-1274,1-1275, or 1-1665 of SEQ ID NO: 9) or carboxy-terminus of the referencepolypeptide (e.g., removing residues 1825-1922, or 1913-1922 of SEQ IDNO: 9), from interal portions of the reference polypeptide (e.g.,removing residues 1787-1799 of SEQ ID NO: 9), or a combination thereof.

A fragment can also be a “functional fragment,” in which case thefragment retains some or all of the activity of the referencepolypeptide as described herein. For example, in some embodiments, afunctional fragment of the polypeptide of SEQ ID NO: 9 can retain someor all of the ability of the polypeptide of SEQ ID NO: 9 to degrade AluRNA. Examples of functional fragments of the polypeptide of SEQ ID NO: 9include the polypeptides of SEQ ID NOS: 10-16. Additional examplesinclude, but are not limited to, the polypeptide of SEQ ID NO: 9,including the following residues: 605-1922, 605-1912, 1666-1922,1666-1912, 605-1786 and 1800-1922, 605-1786 and 1800-1912, 1666-1786 and1800-1922, 1666-1786 and 1800-1912, 1276-1922, 1276-1912, 1276-1786 and1800-1922, 1276-1786 and 1800-1912, 1275-1824, or 1276-1824.

The terms “modified amino acid”, “modified polypeptide”, and “variant”refer to an amino acid sequence that is different from the referencepolypeptide by one or more amino acids, e.g., one or more amino acidsubstitutions. A variant of a reference polypeptide also refers to avariant of a fragment of the reference polypeptide, for example, afragment wherein one or more amino acid substitutions have been maderelative to the reference polypeptide. A variant can also be a“functional variant,” in which the variant retains some or all of theactivity of the reference protein as described herein. The termfunctional variant includes a functional variant of a functionalfragment of a reference polypeptide.

In some embodiments, the DICER Polypeptide can be overexpressed in thecell using a vector comprising a nucleotide encoding the DICERpolypeptide, for example, the nucleotide of SEQ ID NOS: 7 or 8, orappropriate fragment thereof, or a nucleotide encoding a DICERPolypeptide, for example, a nucleotide encoding SEQ ID NOS: 9, 10, 11,12, 13, 14, 15, 16, 18, or 20. As will be recognized by those skilled inthe art, the vector can be a plasmid vector or a viral vector (e.g.,adeno-associated virus, lentivirus, adenovirus.

As noted above, in some embodiments, inhibiting Alu RNA comprises use ofa DICER mRNA. In some embodiments, a functional fragment of a DICER mRNAcould be used. In some embodiments, a DICER mRNA having the sequence ofSEQ ID NOS: 17, 19, or 21, or a functional fragment thereof could beused. In some embodiments an mRNA encoding a DICER Polypeptide could beused, for example, an mRNA encoding SEQ ID NOS: 9, 10, 11, 12, 13, 14,15, 16, 18, or 20.

As noted above, in some embodiments, inhibiting Alu RNA comprisesadministering an oligonucleotide or a small RNA molecule targeting theAlu RNA. Such nucleotides can target and degrade Alu RNA.

As such, in some embodiments, a method is provided includingadministering an oligonucleotide targeting Alu RNA. Examples ofoligonucleotides targeting Alu RNA include those set forth in SEQ IDNOS: 22-26. In some embodiments, more than one oligonucleotide isadministered.

In some embodiments, a method is provided including administering ansiRNA targeting Alu RNA. Examples of siRNAs for targeting Alu RNAinclude those set forth in SEQ ID NOS: 1-6.

The details of one or more embodiments of the presently-disclosedsubject matter are set forth in this document. Modifications toembodiments described in this document, and other embodiments, will beevident to those of ordinary skill in the art after a study of theinformation provided in this document. The information provided in thisdocument, and particularly the specific details of the describedexemplary embodiments, is provided primarily for clearness ofunderstanding and no unnecessary limitations are to be understoodtherefrom. In case of conflict, the specification of this document,including definitions, will control.

While the terms used herein are believed to be well understood by one ofordinary skill in the art, definitions are set forth to facilitateexplanation of the presently-disclosed subject matter.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the presently-disclosed subject matter belongs.Although any methods, devices, and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresently-disclosed subject matter, representative methods, devices, andmaterials are now described.

Following long-standing patent law convention, the terms “a”, “an”, and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a cell” includes aplurality of such cells, and so forth.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as reaction conditions, and so forth usedin the specification and claims are to be understood as being modifiedin all instances by the term “about”. Accordingly, unless indicated tothe contrary, the numerical parameters set forth in this specificationand claims are approximations that can vary depending upon the desiredproperties sought to be obtained by the presently-disclosed subjectmatter.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of in some embodiments ±20%, in someembodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, insome embodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethod.

As used herein, ranges can be expressed as from “about” one particularvalue, and/or to “about” another particular value. It is also understoodthat there are a number of values disclosed herein, and that each valueis also herein disclosed as “about” that particular value in addition tothe value itself. For example, if the value “10” is disclosed, then“about 10” is also disclosed. It is also understood that each unitbetween two particular units are also disclosed. For example, if 10 and15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

The presently-disclosed subject matter is further illustrated by thefollowing specific but non-limiting examples. The following examples mayinclude compilations of data that are representative of data gathered atvarious times during the course of development and experimentationrelated to the present invention.

EXAMPLES

DICER1 reduction in geographic atrophy

In human donor eyes with geographic atrophy (n=10), the presentinventors found using real-time RT-PCR that DICER1 mRNA abundance wasreduced in the macular RPE by 65±3% (mean±SEM; P=0.0036; Mann-Whitney Utest) compared to age-similar human eyes without geographic atrophy(n=11) (FIG. 1 a). Because the best understood function of DICER1 ismiRNA generation³, the present inventors measured the expression ofother enzymes involved in miRNA biogenesis. The abundance of the genesencoding DROSHA or the double stranded RNA (dsRNA) binding proteinDGCR8, which form a complex that processes pri-miRNAs into pre-miRNAs⁵,was not reduced in the RPE of human eyes with geographic atrophy. Therewas also no reduction in the expression of the gene encoding Argonaute 2(AGO2, encoded by EIF2C2), the core component of the miRNA effectorcomplex^(6,7), in the RPE of human eyes with geographic atrophy.Corroborating the mRNA data, the present inventors observed a markedreduction of DICER1 protein expression in the RPE layer of human eyeswith geographic atrophy compared to controls in Western blot (FIG. 1 band FIG. 6) and immunohistochemistry analyses (FIG. 1 c). Interestingly,DICER1 mRNA and protein abundance in the adjacent neural retina wassimilar between the two groups (FIG. 7).

Because DICER1 downregulation is observed in some cell types in cultureconditions in response to various chemical stresses⁸, the presentinventors wondered whether DICER1 reduction in geographic atrophy mightbe a common downstream pathway in dying retina. DICER1 protein levelswere not reduced in the RPE of human eyes with several other retinaldisorders such as vitelliform macular dystrophy, retinitis pigmentosa,or retinal detachment (FIG. 8). Also, Dicer1 mRNA abundance in the RPEin two animal models of retinal degeneration—Ccl2^(−/−) Ccr2^(−/−)(refs. 9,10) and Cp^(−/−) Heph^(−/−) mice¹¹—was unchanged compared totheir background strains (FIG. 8). Gene expression studies in numerousother mouse models of retinal degeneration also have not reportedmodulation of Dicer1 (Supplemental Notes). These data argue that DICER1depletion in the RPE of eyes with geographic atrophy is not a genericresponse of damaged or dying retinal cells in vivo.

DICER1 Depletion Induces RPE Degeneration

To determine the functional consequence of reduced DICER1 levels, thepresent inventors conditionally ablated Dicer1 in mouse RPE cells byinterbreeding “foxed” Dicer1 mice¹² (Dicer1^(f/f)) with BEST1 Cremice¹³, which express Cre recombinase under the control of the RPEcell-specific BEST1 promoter. BEST1 Cre; Dicer1^(f/f) mice uniformlyexhibited dramatic RPE cell degeneration (FIG. 1 d-f) that was evidentby the time of weaning. None of the littermate controls exhibitedsimilar pathology. The present inventors also deleted Dicer1 in adultmouse RPE by subretinal injection of an adeno-associated viral vectorcoding for Cre recombinase under the control of the BEST1 promoter¹⁴(AAV1-BEST1-Cre) in Dicer1^(f/f) mice (FIG. 9). These eyes uniformlydisplayed RPE cell degeneration at 28 days after injection similar tothat observed in mice depleted of Dicer1 expression during development(FIG. 1 g-i; FIG. 9). In contrast, neither the contralateral eyes ofDicer1^(f/f) mice that underwent subretinal injection of AAV1-BEST1-GFPnor the eyes of wild-type mice injected with subretinal AAV1-BEST1-Credeveloped RPE cell degeneration (FIG. 1 g-i and FIG. 9). The RPE celldysmorphology in mice depleted of Dicer1 expression resembled thatobserved in the eyes of humans with RPE atrophy due to AMD (FIG. 10).When cultured RPE cells isolated from Dicer1^(f/f) mice were infectedwith an adenoviral vector coding for Cre recombinase (Ad-Cre), thepresent inventors observed a reduction of cell viability compared toinfection with Ad-Null (FIG. 1 j). Similarly, antisense oligonucleotidemediated knockdown of DICER1 in human RPE cells resulted in increasingcell death over time (FIG. 1 k). Collectively, these data support thehypothesis that DICER1 dysregulation is involved in the pathogenesis ofgeographic atrophy.

DICER1 Depletion Phenotype not Due to miRNA Dysregulation

The present inventors tested whether depletion of other enzymes involvedin miRNA biogenesis also would induce RPE degeneration. Subretinalinjection of AAV1-BEST1-Cre in Drosha^(f/f) (ref. 13), Dgcr8^(f/f)(refs. 15,16), or ¹⁰Ago2^(f/f) mice¹⁷ did not result in the dramatic RPEcell damage that was evident in similarly treated Dicer^(f/f) mice (FIG.11). These data suggest that miRNA imbalances are not responsible forRPE degeneration induced by DICER1 depletion. However, the presentinventors and others have reported^(18,19) that a small subset(approximately 7%) of mammalian miRNAs is generated by Dicer1independent of Drosha and Dgcr8. There is also debate as to whether Ago2is essential for miRNA function: Ago2 deficiency leads to globalreduction of miRNA expression uncompensated by other three Ago proteinsin mice^(17,20) and in mouse embryonic fibroblasts and oocytes^(21,22),yet functional redundancy among Argonaute proteins has been reported inmouse embryonic stem cells²³. The present inventors found no RPEdegeneration in mice deficient in Ago1, Ago3, or Ago4 (FIG. 12). TRBP(the human immunodeficiency virus transactivating response RNA-bindingprotein encoded by Tarbp2) recruits DICER1 to the four Argonauteproteins to enable miRNA processing and RNA silencing (ref 24 and R.Shiekhattar, personal communication); Tarbp2^(−/−) mice too had no RPEdegeneration (FIG. 12). These data suggest that RPE degeneration inducedby Dicer1 ablation involves a mechanism specific to Dicer1 and not tomiRNA machinery in general.

To further investigate whether miRNA imbalances might contribute to thephenotype observed in the setting of DICER1 depletion, the presentinventors studied human HCT116 colon cancer cells in which the helicasedomain in exon 5 of DICER1 was disrupted. Despite the impairment ofmiRNA biogenesis in these HCT-DICER1^(ex5) cells²⁵, there was nodifference between HCT-DICER1^(ex5) and parent HCT116 cells in baselinecell viability (FIG. 13). Collectively, these findings suggest that theprincipal biological effect of DICER1 deficit contributing to thedevelopment of geographic atrophy is not miRNA dysregulation. Thefindings do not, however, exclude the possibility that miRNAdysregulation could promote geographic atrophy through other pathways.

Alu RNA Accumulation in Geographic Atrophy

Because miRNA perturbations could not be implicated, the presentinventors speculated that impaired processing of other dsRNAs might beinvolved. Using an antibody^(26,27) that recognizes long dsRNA (J2), thepresent inventors detected abundant dsRNA immunoreactivity in themacular RPE of human eyes with geographic atrophy (n=10; FIG. 2 a-c). Incontrast, no J2 immunoreactivity was observed in eyes without geographicatrophy (n=10; FIG. 2 d). To identify this dsRNA species, the presentinventors immunoprecipitated RPE lysates with J2 antibody and thensequenced the dsRNA using a T4 RNA ligase-aided, adaptor-based PCRamplification strategy. Interestingly, approximately 300-nt long dsRNAspecies were found in the macular RPE of human eyes with geographicatrophy (12/12) but not in eyes without geographic atrophy (0/18)(P=1.2×10⁻⁸ by Fisher's exact test) (FIG. 2 e).

The present inventors recovered clones from 8 of the 12 geographicatrophy eyes and identified two distinct sequences with high homology(E=3.3×10⁻¹⁰³; 1.1×10⁻⁷⁶) to Alu repeat RNAs (FIG. 14). These sequencesshowed homology to the Alu Sq subfamily consensus sequence. Apart frommitochondrial RNAs that were occasionally found in the RPE of bothgeographic atrophy and normal eyes, Alu RNAs were the only dsRNAtranscripts identified specifically in the geographic atrophy samples.The present inventors confirmed that the J2 monoclonal antibodyrecognized Alu RNA both in immunoblotting and in immunofluorescenceassays (FIG. 15). The present inventors also detected a greater than40-fold increase in the abundance of Alu RNAs in the RPE of human eyeswith geographic atrophy compared to control eyes (n=7), but nosignificant difference in Alu RNA abundance was detected in the adjacentneural retina between the two groups (FIG. 2 f, FIG. 16). The presentinventors did not identify exact matches to these Alu sequences in thereference human genome. This could be attributed to genetic variationsor regions not represented in the reference genome or to chimeric Aluformation. Further studies are needed to elucidate the genomic origin ofand regulatory factors involved in transcription of these Alu RNAs.

DICER1 Depletion Induces Alu RNA Accumulation

The present inventors tested whether Alu RNA accumulation in the RPE ofgeographic atrophy was the result of deficient DICER1 processingactivity. DICER1 knockdown in human RPE cells using antisenseoligonucleotides resulted in increasing Alu RNA accumulation over time(FIG. 3 a, FIG. 17). Similarly, Ad-Cre infection of RPE cells isolatedfrom Dicer1^(f/f) mice resulted in accumulation of B1 and B2 repeat RNAs(FIG. 3 b, c), which are Alu-like short interspersed repetitive elementsin the mouse. Interestingly, DICER1 was expressed in both the nucleusand cytoplasm of RPE cells and its depletion led to accumulation ofAlu/B1/B2 RNA in both cellular compartments (FIG. 3 b-d, FIG. 18). Inaddition, recombinant DICER1 degraded Alu RNA, and the biologicalspecificity of this cleavage was confirmed by the inability ofheat-denatured DICER1 to degrade Alu RNA (FIG. 3 e). Enforced expressionof DICER1 in human RPE cells reduced Alu RNA abundance followingenforced expression of Alu RNA (FIG. 3 f), consistent with degradationof these repetitive transcripts by DICER1 in vivo. Collectively thesedata confirm that DICER1 dysregulation can trigger Alu/B1/B2 RNAaccumulation.

Because cell stresses such as heat shock or viral infection can inducegeneralized retrotransposon activation, the present inventors wonderedwhether Alu RNA accumulation in geographic atrophy might be a genericresponse in dying retina. However, in the RPE of human eyes withgeographic atrophy and in DICER1-depleted human RPE cells, there was noincrease in the abundance of RNAs coded by L1.3 (a long interspersedrepetitive element), human endogenous retrovirus-W envelope (a longterminal repeat retrotransposon), or hY3 (a repetitive small cytoplasmicRo RNA) (FIG. 19). These data demonstrate that Alu RNA accumulation is abiologically specific response to DICER1 depletion.

To determine whether Alu RNA accumulation was derived from RNApolymerase II (Pol II) or Pol III transcription, the present inventorsperformed experiments using α-amanitin (a Pol II inhibitor) andtagetitoxin (a Pol III inhibitor). Alu RNA upregulation induced byDICER1 knockdown was inhibited by tagetitoxin but not α-amanitin (FIG.20). The present inventors also found using Northern blotting that AluRNA from the RPE of human eyes with geographic atrophy was approximately300 nucleotides in length, consistent with the length of non-embeddedPol III Alu transcripts. Because homology between Alu RNA and 7SL RNA,the evolutionary precursor of Alu, can complicate interpretation ofnorthern blot analysis, the present inventors reprobed these samplesusing a probe that specifically detects the non-Alu “S domain” of 7SLRNA. In contrast to the increased amounts of RNA species detected by theAlu-targeting probe in geographic atrophy RPE, there was no differencein 7SL RNA abundance. The present inventors also confirmed that the Aluprobe did not detect endogenous 7SL RNA under the stringent conditionsthe present inventors employed. Corroborating these data, real-timeRT-PCR analysis showed that 7SL RNA was not dysregulated in the RPE ofhuman eyes with geographic atrophy or in DICER1-depleted human RPE cells(FIG. 21).

DICER1 knockdown also did not induce upregulation of several PolII-transcribed genes (ADAR2, NICN, NLRP, SLFN 11) that contain embeddedAlu sequences in their exons. Collectively, these data suggest that AluRNA detected in the RPE of human eyes with geographic atrophy areprimary Alu transcripts and not passenger or bystander sequencesembedded in other RNAs. Conclusive assignment of these Alu sequences asPol III transcripts must await precise determination of theirtranscription start site.

Alu RNA Induces RPE Degeneration

Next the present inventors tested whether accumulation of Alu RNA mightpromote the development of geographic atrophy. Transfecting human orwild-type mouse RPE cells with a plasmid coding for Alu (pAlu) reducedcell viability (FIG. 4 a). Subretinal transfection of plasmids codingfor two different Alu RNAs or for B1 or B2 RNAs induced RPE degenerationin wild-type mice (FIG. 4 b, FIG. 22, and data not shown). Treatment ofhuman RPE cells with a recombinant 281 nucleotide (nt)-long Alu RNA thatis identical to a Pol III derived Alu RNA isolated from a humanembryonal carcinoma cell line, i.e., a single RNA strand that folds intoa defined secondary structure, resulted in a dose-dependent increase incell death (FIG. 4 c). These findings suggest that endogenous DICER1 candegrade small amounts of Alu RNA but are overwhelmed by high levels.Consistent with this concept, overexpression of DICER1 blockedpAlu-induced cell death in human RPE cells (FIG. 4 d) and RPEdegeneration in wild-type mice (FIG. 4 e).

The present inventors verified that subretinal injection of Alu RNAresulted in its delivery to RPE cells in wild-type mice (FIG. 23),consistent with the ability of long RNAs with duplex motifs to entercells²⁸. The present inventors then cloned a 302-nt long Alu RNAisolated from the RPE of a human eye with geographic atrophy andtranscribed it in vitro to generate partially and completely annealedstructures that mimic Alu RNAs transcribed by Pol III and Pol II,respectively. Subretinal injection of either of these Alu RNAs resultedin RPE degeneration in wild-type mice (FIG. 4 f, FIG. 24), supportingthe assignment of disease causality in accord with the molecular Koch'spostulates. In contrast, subretinal injection of these Alu RNAs digestedwith DICER1 did not induce RPE degeneration in wild-type mice (FIG. 4 g,FIG. 24). When these Alu RNAs were subjected to mock DICER1 digestion,they retained their ability to induce RPE degeneration, suggesting arole for DICER1 in protecting against Alu RNA-induced degeneration.

The present inventors tested whether other structured RNAs of similarlength as Alu would damage the retina. Subretinal transfection oftransfer RNA or plasmids coding for 7SL RNA or two different primarymiRNAs did not induce RPE degeneration in wild-type mice (FIG. 25). Thepresent inventors reported that chemically synthesized dsRNAs that mimicviral dsRNA can induce RPE degeneration by activating toll likereceptor-3 (TLR3)²⁹, a pattern receptor that generically recognizesdsRNA. However, transfection of a plasmid coding for Alu RNA did notinduce TLR3 phosphorylation in human RPE cells and did induce RPEdegeneration in Tlr3^(−/−) mice (FIG. 26). These results indicate thatthe ability of Alu RNA to induce RPE degeneration cannot be attributedsolely to its repetitive or double stranded nature, as it exertedeffects distinct from other structured dsRNAs of similar length.

The mechanism of RPE cell death in geographic atrophy has not beenpreviously defined. DNA fragmentation has been identified in RPE cellsin human eyes with geographic atrophy³⁰, and Dicer1 knockdown has beenassociated with induction of apoptosis in diverse tissues^(12,31). Thepresent inventors now provide evidence of caspase-3 cleavage in regionsof RPE degeneration in human eyes with geographic atrophy (FIG. 27).Caspase-3 cleavage was also observed in the RPE cells of BEST1Cre;Dicer1^(f/f) mice and in Alu RNA-stimulated or -overexpressing human RPEcells. These data suggest a role for Alu RNA-induced RPE cell apoptosistriggered by DICER1 dysregulation in geographic atrophy.

Although the present inventors show that Alu RNA induces RPEdegeneration, the presented observations could be consistent with theidea that an imbalance in small RNA species produced from long Alu RNAscould contribute to the RPE degeneration phenotype. To study thisquestion, the present inventors exposed human RPE cells or wild-typemice to DICER1 cleavage fragments of Alu RNA. Subretinal transfection ofthese fragments alone in wild-type mice had no detectable effect on RPEcell morphology, and co-administering these fragments did not preventRPE cell degeneration induced by subretinal transfection of a plasmidcoding for Alu RNA (FIG. 28). Similarly, these fragments did not preventhuman RPE cell death induced by overexpression of Alu RNA. These datasuggest that upregulation of long Alu RNA rather than imbalance in AluRNA-derived small RNA fragments is responsible for RPE degenerationinduced by DICER1 reduction.

As these experiments were performed with in vitro cleavage fragments thepresent inventors cannot be certain whether in vivo cleavage fragmentswould function similarly. However, Alu RNAs with varying sequencesinduced RPE degeneration in vivo. Because the cleavage fragments ofthese different Alu RNAs would not be identical it is unlikely that theyall execute identical biological functions, particularly if theyfunctioned as miRNAs. Another line of evidence that Alu RNA, and not itscleavage fragments, is responsible for RPE degeneration comes fromfunctional rescue experiments (see below) wherein antisense-mediatedinhibition of Alu RNA blocks human RPE cell death induced by DICER1knockdown and inhibition of B1/B2 RNA blocks RPE degeneration inDicer1-depleted mice and mouse RPE cells. Because these antisensetreatments would not be expected to alter the reduced levels ofDICER1-cleaved Alu/B1/B2 RNA fragments, the imbalance in these fragmentsis unlikely to have induced RPE degeneration. Nevertheless, subtlefunctions of these small RNAs in modulating Alu RNA induced pathologycannot be excluded.

To dissect the contribution of Alu RNA accumulation versus that of miRNAdysregulation to RPE degeneration in the context of reduced DICER1expression, the present inventors re-examined HCT-DICER1^(ex5) cells inwhich miRNA biogenesis is impaired but long dsRNA cleavage is preserveddue to the intact RNase III domains. The present inventors found nosignificant difference in Alu RNA levels between HCT-DICER1^(ex5) andparent HCT116 cells (FIG. 29). In contrast, when DICER1 was knocked downby antisense oligonucleotides in HCT116 cells, increased Alu RNAaccumulation was observed. Also, Alu RNA induces similar levels ofcytotoxicity in HCT-DICER1^(ex5) and parent HCT116 cells, suggestingthat coexisting miRNA expression deficits do not augment Alu RNA inducedRPE degeneration. In conjunction with the discordance in the RPEdegeneration phenotype between ablation of Dicer1 and that of variousother small RNA biogenesis pathway genes in mice, the findings suggestthat Alu RNA accumulation is critical to cytotoxicity induced by DICER1reduction.

RPE Degeneration Blocked by Alu RNA Inhibition

The present inventors then tested whether the cytotoxic effects ofDICER1 reduction could be attributed to Alu RNA accumulation. DICER1knockdown in human RPE cells by antisense oligonucleotides reduced cellviability (FIG. 5 a). This cytotoxic effect of DICER1 reduction wasinhibited by antisense oligonucleotides targeting Alu RNA sequences butnot by a scrambled antisense control (FIG. 5 a, b and FIG. 21). Ad-Creinfection of RPE cells isolated from Dicer1^(f/f) mice resulted inreduced cell viability, and this was blocked by antisenseoligonucleotides targeting both B1 and B2 repeat RNAs but not by ascrambled antisense control (FIG. 5 c, d). Subretinal administration ofantisense oligonucleotides that reduced accumulation of B1 and B2 RNAsalso inhibited RPE degeneration in Dicer1^(f/f) mice treated withAAV1-BEST1-Cre (FIG. 5 e, f), providing evidence of in vivo functionalrescue.

The present inventors tested whether Alu inhibition also rescued miRNAexpression deficits as a potential explanation for the functional rescueof RPE degeneration induced by DICER1 depletion. As expected, DICER1knockdown in human RPE cells reduced the abundance of numerous miRNAsincluding let-7a, which is ubiquitously expressed, miR-184, miR-204/211,and miR-221/222, which are enriched in the RPE, and miR-320a, andmiR-484 and miR-877, which are DROSHA/DGCR8-independent andDICER1-dependent (FIG. 5 g). However, inhibition of Alu RNA did not leadto recovery of miRNA expression in these DICER1-depleted cells. Thus therescue of RPE cell viability by Alu RNA inhibition despite thepersistence of global miRNA expression deficits argues that RPEdegeneration induced by DICER1 deficit is due to Alu RNA accumulationand not miRNA dysregulation.

These data, taken together, support a model in which primary Alutranscripts are responsible for the observed RPE degeneration. Whethersimilar pathology can also result from upregulation of as yet undefinedPol II transcripts with embedded Alu sequences is an intriguingpossibility that may be addressed in future studies. Importantly, thepresent inventors show here that primary Alu transcripts are elevated inhuman disease, that Alu transcripts recapitulate disease in relevantexperimental models, and that targeted suppression of Alu transcriptssuccessfully inhibits this pathology. These observations have directrelevance for clinical strategies to prevent and treat geographicatrophy.

DISCUSSION

The findings elucidate a critical cell survival function for DICER1 byfunctional silencing of toxic Alu transcripts. This unexpected functionsuggests that RNAi-independent mechanisms should be considered ininterpreting the phenotypes of systems in which Dicer1 is dysregulated.For example, it would be interesting to test the speculation that Dicer1ablation induced cell death in mouse neural retina³² and heart³³ mightalso involve B1/B2 RNA accumulation. More broadly, recognition of DICER1's hitherto unidentified function as an important controller oftranscripts derived from the most abundant repetitive elements in thehuman and mouse genomes can illuminate new functions for RNases incytoprotective surveillance. DICER1 expression is reduced in geographicatrophy and partial loss of DICER promotes RPE degeneration; thus thepresent inventors could speculate that loss of heterozygosity in DICER1may underlie the development of geographic atrophy, similar to itsfunction as a haploinsufficient tumor suppressor³⁴⁻³⁶.

This also is, to our knowledge, the first example of how Alu could causea human disease via direct RNA cytotoxicity rather than by inducingchromosomal DNA rearrangements or insertional mutagenesis throughretrotransposition, which have been implicated in diseases such asα-thalassemia³⁷, colon cancer³⁸, hypercholesterolemia^(39,40), andneurofibromatosis⁴¹. Future studies can be employed to determine theprecise chromosomal locus of the Alu RNA elements that accumulate ingeographic atrophy and the nature of transcriptional andpost-transcriptional machinery that enable their biogenesis.

In addition to processing miRNAs³, DICER1 has been implicated inheterochromatin assembly^(42,43.) Since Alu repeat elements are abundantwithin heterochromatin⁴⁴, it would be interesting to investigate whetherperturbations in centromeric silencing also underlie the pathogenesis ofgeographic atrophy. Indeed, the finding that chromatin remodelling atAlu repeats can regulate miRNA expression⁴⁵ raises the intriguingpossibility of other types of regulatory intersections between DICER1and Alu. It also remains to be investigated whether centromericsatellite repeats that have been described to accumulate in Dicer1-nullmouse embryonic stem cells^(46,47) might be involved in the pathogenesisof geographic atrophy.

In the mouse germline, Dicer1 has been implicated in the generation ofendogenous small interfering RNAs (endo-siRNAs) from repeatelements^(48,49). If this process is conserved in mammalian somatictissues, it would be interesting to learn whether endo-siRNAs serve ahomeostatic function in preventing the development of geographicatrophy. A recent study in nematodes demonstrated that caspases cancleave Dicer1 and convert it into a DNase that promotes apoptosis⁵⁰. Thefinding that Alu RNA can induce caspase activation therefore introducesthe possibility of bidirectional regulation between DICER1 and Alu thatcould trigger feed-forward loops that further amplify the disease state.

The inciting events that trigger an RPE-specific reduction of DICER1 inpatients with geographic atrophy remain to be determined. Potentialculprit could include oxidative stress, which is postulated to underlieAMD pathogenesis², as the present inventors found that exposure tohydrogen peroxide downregulates DICER1 in human RPE cells (FIG. 30).While the upstream triggers of DICER1 dysregulation and the possiblerole of other DICER-dependent, DROSHA/DGCR8-independent small RNAs ingeographic atrophy await clarification, the ability of Alu RNA antisenseoligonucleotides to inhibit RPE cell death induced by DICER1 depletionprovides a rationale to investigate Alu RNA inhibition or DICER1augmentation as potential therapies for geographic atrophy.

Additional Notes

Dicer1 mRNA levels are not modulated in multiple mouse models of retinaldegeneration including light damage^(53,54), hyperoxia, ⁵⁵retinaldetachment^(53,56), Crx^(−/−) mice⁵⁷, Rslh^(−/−) mice⁵⁸, rd1mice^(59,60), cpfl1 mice⁶¹, or Mitf mice⁶². Dicer1 abundance also is notreduced in mouse models of cellular stress in the retina includingexposure to advanced glycation endproducts⁶³ or retinal detachment⁶⁴.Therefore, Dicer1 downregulation is not a generic late-stage stressresponse in the retina.

Materials and Methods

Animals

All animal experiments were approved by institutional review committeesand the Association for Research in Vision and Ophthalmology. C57Bl/6Jand Dicer1^(f/f) mice were purchased from The Jackson Laboratory.Transgenic mice that express Cre recombinase in the retinal pigmentedepithelium under the control of the human bestrophin-1 promoter (BEST1Cre mice), DGCR8^(f/f), Drosha^(f/f), Tarbp2^(−/−), Ccl2^(−/−)Ccr2^(−/−), and Cp^(−/−) Heph^(−/−) mice have been previouslydescribed⁶⁵⁻⁷¹. Ago2^(f/f) mice⁷² and mice deficient in Ago1, Ago3, orAgo4 (ref. 73) were generously provided by A. Tarakhovsky. For allprocedures, anaesthesia was achieved by intraperitoneal injection of 50mg/kg ketamine hydrochloride (Ft. Dodge Animal Health) and 10 mg/kgxylazine (Phoenix Scientific), and pupils were dilated with topical 1%tropicamide (Alcon Laboratories).

Fundus Photography.

Retinal photographs of dilated mouse eyes were taken with a TRC-50 IXcamera (Topcon) linked to a digital imaging system (Sony).

Human Tissue.

Donor eyes or ocular tissues from patients with geographic atrophy dueto AMD or patients without AMD were obtained from various eye banks inAustralia and the United States of America. These diagnoses wereconfirmed by dilated ophthalmic examination prior to acquisition of thetissues or eyes or upon examination of the eye globes post mortem. Thestudy followed the guidelines of the Declaration of HelsinkiInstitutional review boards granted approval for allocation andhistological analysis of specimens.

Immunolabeling.

Human eyes fixed in 2-4% paraformaldehyde were prepared as eyecups,cryoprotected in 30% sucrose, embedded in optimal cutting temperaturecompound (Tissue-Tek OCT; Sakura Finetek), and cryosectioned into 10 μmsections. Depigmentation was achieved using 0.25% potassium permanganateand 0.5% oxalic acid. Immunohistochemical staining was performed withthe mouse antibody against dsRNA (1:1,000, clone J2, English &Scientific Consulting) or rabbit antibody against human DICER1 (1:100,Santa Cruz Biotechnology). Isotype IgG was substituted for the primaryantibody to assess the specificity of the staining Bound antibody wasdetected with biotin-conjugated secondary antibodies (VectorLaboratories). Slides were further incubated in alkalinephosphatase-streptavidin solution (Invitrogen) and the enzyme complexwas visualized by Vector Blue (Vector Laboratories). Levamisole (VectorLaboratories) was used to block endogenous alkaline phosphataseactivity. Slides were washed in PBS, rinsed with deionized water,air-dried, and then mounted in Clear Mount (EMS). Mouse RPE/choroid flatmounts were fixed with 4% paraformaldehyde or 100% methanol and stainedwith rabbit antibodies against human zonula occludens-1 (1:100,Invitrogen), Cre recombinase (1:1000, EMD4Biosciences), or human cleavedcaspase-3 (1:200, Cell Signaling) and visualized with Alexa594- orCy5-conjugated secondary antibodies. Both antibodies are cross-reactiveagainst the mouse homologues. Primary human RPE cells were grown to70-80% confluency in chamber slides (Lab-Tek). After 24 h oftransfection with pAlu or pUC19, cells were fixed in acetone for 10 minat −20° C. Cells were blocked with PBS-3% BSA and incubated with mouseantibody against dsRNA (1:500, clone J2) overnight at 4° C. andvisualized with Alexa Fluor 488-conjugated secondary antibodies. ForDICER1 staining, cells were fixed in methanol/acetone (7:3) for 30 minon ice, blocked with PBS-3% BSA-5% FBS, incubated with rabbit antibodyagainst human DICER1 (1:100, Santa Cruz Biotechnology) overnight at 4°C., and visualized with goat-anti-rabbit Alexa Fluor 594-conjugatedsecondary antibodies. After DAPI counterstaining, slides were coverslipped in Vectashield (Vector Laboratories). Images were obtained usingthe Leica SP-5 or Zeiss Axio Observer Z1 microscopes.

Histology.

Mouse eyes were fixed with 4% paraformaldehyde and 3.5% glutaraldehyde,postfixed in 2% osmium tetroxide, and dehydrated in ethanol andembedded. Semi-thin (1 μm) sections were cut and stained with toluidineblue. Bright field images were obtained using the Zeiss Axio Observer Z1microscope.

Subretinal Injection.

Subretinal injections (1 μL) in mice were performed using aPico-Injector (PLI-100, Harvard Apparatus). In vivo transfection ofplasmids coding for DICER1 (ref. 74), Alu Ya5 (ref. 75), Alu Yb9 (ref.76), 7SL RNA (ref. 77), pri-miR29b1 (Addgene), or pri-miR26a2 (Addgene)and bovine tRNA (Sigma-Aldrich) (0.5 mg/mL) was achieved using 10%Neuroporter (Genlantis). AAV1-BEST1-Cre⁷⁸ or AAV1-BEST1-GFP wereinjected at 1.0×10¹¹ pfu/mL and recombinant Alu RNAs (1: a single RNAstrand of 281 nucleotides whose sequence is that of the cDNA clone TS103 (ref 51) and folds into a defined secondary structure identical to aPol III derived transcript; 2: a single RNA strand of 302 nucleotideswhose sequence is identical to that of a clone isolated from the RPE ofa human eye with geographic atrophy that folds into a defined secondarystructure identical to a Pol III derived transcript; or 3: a fullycomplementary dsRNA version of this 302 nucleotide long sequence thatmimics a Pol II derived transcript) was injected at 0.3 mg/mL.Cell-permeating cholesterol conjugated-B1/B2 antisense oligonucleotides(as) (5′-TCAGATCTCGTTACGGATGGTTGTGA-3′) or cholesterolconjugated-control as (5′-TTGGTACGCATACGTGTTGACTGTGA-3′) (both fromIntegrated DNA Technologies) were injected (2 μg in 1 μL) 10 days afterAAV1-BEST1-Cre was injected in Dicer1^(f/f) mice.

Isolation of dsRNA.

Human eyes were stored in RNAlater (Ambion). Tissue extracts wereprepared by lysis in buffer containing 50 mM Tris-HCl, pH 8, 150 mMNaCl, 1% Nonidet P-40, protease and phosphatase inhibitors (completemini EDTA-free, protease inhibitor and phosphatase inhibitor cocktailtablets, Roche), and RNase inhibitor (SUPERase-In, Ambion). Afterhomogenization using bullet blender (Nextadvance) and centrifugation,immunoprecipitations were performed by adding 40 μg of mouse antibodyagainst dsRNA (clone J2) for 16 h at 4° C. Immunocomplexes werecollected on protein A/G agarose (Thermoscientific) and dsRNA specieswere separated and isolated using Trizol (Invitrogen) according to themanufacturer's instructions.

Ligation of dsRNA and Anchor Primer.

An anchor primer (PC3-T7 loop,5′-p-GGATCCCGGGAATTCGGTAATACGACTCACTATATTTTTATAGTGAGTCGTATTA-OH-3′,200-400 ng, IDT)^(79,80) was ligated to dsRNA (200-400 ng) in 50 mMHEPES/NaOH, pH 8 (vWR), 18 mM MgCl₂ (Invitrogen), 0.01% BSA (FisherScientific), 1 mM ATP (Roche), 3 mM DTT (Fluka), 10% DMSO (Finnzymes),20% PEG 6000 (Alfa Aesar), and 30U T4 RNA ligase (Ambion). Ligation wasperformed at 37° C. for 16 h, and ligated dsRNA was purified by MinEluteGel extraction columns (Qiagen).

Sequence-Independent cDNA Synthesis.

After denaturation, ligated dsRNA was reverse transcribed in a RTreaction containing 50 mM Tris-HCl, pH 8.3, 10 mM MgCl₂, 70 mM KCl, 30mM β-mercaptoethanol, 1 mM dNTPs and 15U cloned AMV reversetranscriptase (Invitrogen). The mixture was incubated in a thermalcycler (Eppendorf) at 42° C. for 45 min followed by 55° C. for 15 min.

Polymerase Chain Reaction (PCR) Amplification.

Amplification of cDNA was performed using primer PC2(5′-p-CCGAATTCCCGGGATCC-3′, IDT) in a reaction buffer containing 5 μLcDNA and 40 μL Platinum PCR SuperMix (Invitrogen). The PCR cyclingparameters consisted of one step of 72° C. for 1 min to fill incompletecDNA ends and produce intact DNA, followed by one step of initialdenaturation (94° C., 2 min), 39 cycles of 94° C. for 30 s, 53° C. for30 s, and 72° C. for 1 min, and a final extension step of 72° C. for 10min. In vitro transcribed dsRNAs of varying lengths (325 bp, 1 and 2 kb)were used as positive controls.

Cloning and Sequencing.

The amplified cDNA products were incubated with 1U calf intestinalalkaline phosphatase (Invitrogen) at 37° C. for 5 min to remove the5′-phosphate group, separated on a low-melting point agarose gel (1%)and purified using Qiaquick gel extraction kit (Qiagen). The purifieddephosphorylated cDNA fragments were cloned in PCRII TOPO vector(Invitrogen) and sequenced using M13 forward (−20) and M13 reverseprimers at the University of Kentucky Advanced Genetic TechnologiesCenter using multi-colour fluorescence based DNA sequencer (ABI 3730x1).Sequences were assembled using ContigExpress from vector NTI Advance.The homology of the isolated cDNA sequences to known Alu consensussequences was determined using the CENSOR server⁸¹ (a WU-BLAST-powereddatabase of repetitive elements (http://www.girinst.org/censor). Foreach cDNA sequence, the homologous region of the query was aligned tothe consensus Alu sequence using BLASTn⁸²(http://www.ncbi.nlm.nih.gov/BLAST). Multiple sequence alignment wasperformed using ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2). Theconsensus sequences have been deposited in GenBank under the accessionnumbers HN176584 and HN176585.

Alu RNA Synthesis.

The present inventors synthesized two Alu RNAs: a 281 nt Alu sequenceoriginating from the cDNA clone TS 103 which is known to be expressed inhuman cells⁵¹ and a 302 nt Alu sequence isolated from the RPE of a humaneye with geographic atrophy. Both of these Alu RNAs were synthesizedusing a RNA polymerase T7 promoter and runoff transcription followed bygel purification as previously described⁸³. This yields single strandedRNAs that fold into a defined secondary structure identical to Pol IIIderived transcripts. The present inventors also synthesized a fullycomplementary dsRNA form (resembling a Pol II derived transcript) of the302 nt human geographic atrophy Alu using linearized PCRII TOPO plasmidtemplates using T7 or SP6 RNA polymerases (MegaScript, Ambion) accordingto the manufacturer's recommendations. After purification, equal molaramount of each transcript were combined and heated at 95° C. for 1 min,cooled and then annealed at room temperature for 24 h. The Alu dsRNA wasprecipitated, suspended in water and analyzed on 1.4% non-denaturingagarose gel using the single-stranded complementary strands as controls.

Real-Time PCR.

Total RNA was extracted from tissues or cells using Trizol reagent(Invitrogen) according to manufacturer's recommendations and weretreated with RNase free DNase (Ambion). Total RNA (1 μg) was reversetranscribed as previously described⁷⁰ using qScript cDNA SuperMix(Quanta Biosciences). The RT products (cDNA) were amplified by real-timequantitative PCR (Applied Biosystems 7900 HT Fast Real-Time PCR system)with Power SYBR green Master Mix. Oligonucleotide primers specific forDICER1 (forward 5′-CCCGGCTGAGAGAACTTACG-3′ and reverse5′-CTGTAACTTCGACCAACACCTTTAAA-3′), DROSHA (forward5′-GAACAGTTCAACCCCGATGTG-3′ and reverse 5′-CTCAACTGTGCAGGGCGTATC-3′),DGCR8 (forward 5′-TCTGCTCCTTAGCCCTGTCAGT-3′ and reverse5′-CCAACACTCCCGCCAAAG-3′), EIF2C2 (forward 5′-GCACGGAAGTCCATCTGAAGTC-3′and reverse 5′-CCGGCGTCTCTCGAGATCT-3′), human 18S rRNA (forward5′-CGCAGCTAGGAATAATGGAATAGG-3′ and reverse 5′-GCCTCAGTTCCGAAAACCAA-3′),Alu (forward 5′-CAACATAGTGAAACCCCGTCTCT-3′ and reverse5′-GCCTCAGCCTCCCGAGTAG-3′), LINE L1.3 (ORF2) (forward5′-CGGTGATTTCTGCATTTCCA-3′ and reverse 5′-TGTCTGGCACTCCCTAGTGAGA-3′),HERV-WE1 (forward 5′-GCCGCTGTATGACCAGTAGCT-3′ and reverse5′-GGGACGCTGCATTCTCCAT-3′), human Ro-associated Y3 (hY3) (forward5′-CCGAGTGCAGTGGTGTTTACA-3′ and reverse5′-GGAGTGGAGAAGGAACAAAGAAATC-3′), 7SL (forward5′-CGGCATCAATATGGTGACCT-3′ and reverse 5′-CTGATCAGCACGGGAGTTTT-3′), B1(forward 5′-TGCCTTTAATCCCAGCACTT-3′ and reverse5′-GCTGCTCACACAAGGTTGAA-3′), B2 (forward 5′-GAGTTCAAATCCCAGCAACCA-3′ andreverse 5′-AAGAGGGTCTCAGATCTTGTTACAGA-3′), cytoplasmic B2 (forward5′-GCCCTGTTACAATTGGCTTT-3′ and reverse 5′-GTGGTTGCTGGGATTTGAAC-3′),

Dicer1 (forward 5′-CCCACCGAGGTGCATGTT-3′ and reverse5′-TAGTGGTAGGAGGCGTGTGTAAAA-3′), mouse 18S rRNA (forward5′-TTCGTATTGCGCCGCTAGA-3′ and reverse 5′-CTTTCGCTCTGGTCCGTCTT-3′) wereused. The QPCR cycling conditions were 50° C. for 2 min, 95° C. for 10min followed by 40 cycles of a two-step amplification program (95° C.for 15 s and 58° C. for 1 min). At the end of the amplification, meltingcurve analysis was applied using the dissociation protocol from theSequence Detection system to exclude contamination with unspecific PCRproducts. The PCR products were also confirmed by agarose gel and showedonly one specific band of the predicted size. For negative controls, noRT products were used as templates in the QPCR and verified by theabsence of gel-detected bands. Relative expressions of target genes weredetermined by the 2^(−ΔΔCt) method.

miRNA PCR.

miRNA abundance was quantified using the All-in-One™ miRNA qRT-PCRDetection Kit (GeneCopoeia). Briefly, total RNA was polyadenylated andreverse transcribed using a poly dT-adaptor primer. Quantitative RT-PCRwas carried out using a miRNA-specific forward primer and universalreverse primer. PCR products were subjected to dissociation curve andgel electrophoresis analyses to ensure that single, mature miRNAproducts were amplified. Data were normalized to ACTB levels. Theforward primers for the miRNAs were as follows: miR-184(5′-TGGACGGAGAACTGATAAGGGT-3′); miR-221/222(5′-AGCTACATCTGGCTACTGGGT-3′); miR-204/211(5′-TTCCCTTTGTCATCCTTCGCCT-3′); miR-877 (5′-GTAGAGGAGATGGCGCAGGG-3′);miR-320a (5′-AAAAGCTGGGTTGAGAGGGCGA-3′); miR-484(5′-TCAGGCTCAGTCCCCTCCCGAT-3′); let-7a (5′-TGAGGTAGTAGGTTGTATAGTT-3′).The reverse primers were proprietary (Genecopoeia). The primers for ACTBwere forward (5′-TGGATCAGCAAGCAGGAGTATG-3′) and reverse(5′-GCATTTGCGGTGGACGAT-3′).

Dot Blot (Immuno-Dot Binding).

Increasing amounts of Alu RNA were spotted onto hybond-N⁺ positivelycharged nylon membrane (Amersham) and UV cross-linked. After blocking,the membranes were incubated with mouse antibody against dsRNA (1:1,000,clone J2) for 1 h at RT. The peroxidase-conjugated goat anti-mousesecondary antibody (1:5,000, Sigma) was used for 1 h at RT. Afterseveral washes, the signals were visualized by enhancedchemiluminescence (ECL plus, Amersham). In vitro transcribed dsRNAs ofdifferent length were used as positive controls. Transfer and ribosomalRNAs were used as negative controls.

Northern Blot.

Total RNA from normal and diseased macular RPE was extracted asdescribed above using Trizol. RNA integrity and quality was assessedusing 1% agarose gel electrophoresis and RNA concentrations and puritywere determined for each sample by NanoDrop 1000 spectrophotometer V3.7(Thermo Fisher Scientific). dsRNA (2 μg) was separated on denaturing 15%PAGE-urea ready gel (Bio-Rad), and total RNA (10 μg) was separated bysize on 1% agarose, 0.7M formaldehyde gels and visualized on anultraviolet transilluminator to ensure consistent loading betweendifferent groups and to record the distance of migration of the 18S and28S rRNA bands. dsRNA ladder (21-500 bp, New England BioLabs) and RNAladder (0.1-2 kb, Invitrogen) were used as markers. Gels were thentransferred to a positively charged Nylon membrane (Hybond-N+, GEHealthcare Bio-Sciences) by vacuum blotting apparatus (VacuGene XLVacuum Blotting System, GE Healthcare Bio-Sciences). The RNAs werecrosslinked to the membranes by ultraviolet irradiation and baked at 80°C. for 20-30 min. Membranes were hybridized with (α-³²P)-dCTP-labeledDNA Alu probe at 42° C. overnight. On the following day, the membraneswere rinsed twice with 1×SSC, 0.1% SDS at 55° C. Each wash was for 20min, and then membranes were subjected to storage in a phosphorautoradiography cassette. Hybridization signals were determined by usingTyphoon phosphorimager (GE Healthcare Bio-Sciences). The 7SL probe wassynthesized by PCR amplification of a 7SL RNA plasmid^(77,84) with thefollowing primers (forward 5′-ATCGGGTGTCCGCACTAAG-3′ and reverse5′-ATCAGCACGGGAGTTTTGAC-3′) designed to amplify a 128-bp fragment withinthe S-region that is not contained in Alu. For visualization of U6,membranes were stripped and blotted again using the High Sensitive MiRNANorthern Blot Assay Kit (Signosis) according to the manufacturer'sinstructions.

Western Blot.

Tissues were homogenized in lysis buffer (10 mM Tris base, pH 7.4, 150mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.5% NP-40, protease andphosphatase inhibitor cocktail (Roche)). Protein concentrations weredetermined using a Bradford assay kit (Bio-Rad) with bovine serumalbumin as a standard. Proteins (40-100 μg) were run on 4-12% NovexBis-Tris gels (Invitrogen). The transferred membranes were blocked for 1h at RT and incubated with antibodies against DICER1 (1:1,000, ref. 85;or 1:200, Santa Cruz Biotechnology) at 4° C. overnight. Protein loadingwas assessed by immunoblotting using an anti-Tubulin antibody (1:1,000;Sigma-Aldrich). The secondary antibodies were used (1:5,000) for 1 h atRT. The signal was visualized by enhanced chemiluminescence (ECL Plus)and captured by VisionWorksLS Image Acquisition and Analysis software(Version 6.7.2, UVP, LLC). Densitometry analysis was performed usingImageJ (NIH). The value of 1 was arbitrarily assigned for normal eyesamples.

DICER1 Cleavage.

The ability of DICER1 to cleave Alu RNA was tested using RecombinantHuman Dicer Enzyme Kit (Genlantis) according the manufacturer'sinstructions. The products of the digestion were purified for the invivo injection using RNA Purification Column (Genlantis).

Cell Culture.

All cell lines were cultured at 37° C. and 5% CO₂. Primary mouse RPEcells were isolated as previously described⁸⁶ and grown in DulbeccoModified Eagle Medium (DMEM) supplemented with 10% FBS and standardantibiotics concentrations. Primary human RPE cells were isolated aspreviously described⁸⁷ and maintained in DMEM supplemented with 20% FBSand antibiotics. Parental HCT116 and isogenic Dicer^(ex5) cells²⁵ werecultured in McCoy's 5A medium supplemented with 10% FBS.

Transient Transfection.

Human and mouse RPE cells were transfected with pUC19, pA1u,pCDNA3.1/Dicer1-FLAG, pCDNA3.1, DICER1 antisense oligonucleotide (as)(5′-GCUGACCTTTTTGCTUCUCA-3′), B1/B2 as(5′-TCAGATCTCGTTACGGATGGTTGTGA-3′), control (for DICER1 and B1/B2) as(5′-TTGGTACGCATACGTGTTGACTGTGA-3′), Alu as(5′-CCCGGGTTCACGCCATTCTCCTGCCTCAGCCTCACGAGTAGCTGGGACTACAGGCGCCCGACACCACTCCCGGCTAATTTTTTGTATTTTT-3′), control (for Alu) as(5′-GCATGGCCAGTCCATTGATCTTGCACGCTTGCCTAGTACGCTCCTCAACCTATCCTCCTAGCCCGTTACTTGGTGCCACCGGCG-3′) using Lipofectamine 2000 (Invitrogen) orOligofectamine (Invitrogen) according to the manufacturer'sinstructions.

Adenoviral Infection.

Cells were plated at density of 15×10³/cm² and after 16 h, atapproximately 50% confluence, were infected with AdCre or AdNull (VectorLaboratories) with a multiplicity of infection of 1,000.

RNA Polymerase Inhibition.

Human RPE cells were transfected with DICER1 or control antisenseoligonucleotides using Lipofectamine 2000. After a change of medium at 6h, the cells were incubated with 45 μM tagetitoxin (EpicentreTechnologies, Tagetin) or 10 μg/ml a-amanitin (Sigma-Aldrich) and thetotal RNA was collected after 24 h.

Cell Viability.

MTS assays were performed using the CellTiter 96 A Queous One SolutionCell Proliferation Assay (Promega) in according to the manufacturer'sinstructions.

Caspase-3 Activity.

Sub-confluent human RPE cells were treated with PBS or Alu RNA atdifferent concentrations in 2% FBS medium for 8 h. The caspase-3activity was measured using Caspase-3 Fluorimetric Assay (R&D Systems)according to the manufacturer's instructions.

Oxidative Stress.

Confluent human RPE cells were exposed to hydrogen peroxide (0-2 mM,Fisher Scientific).

Statistics.

Results are expressed as mean±SEM, with P<0.05 considered statisticallysignificant. Differences between groups were compared by usingMann-Whitney U test or Student t test, as appropriate, and 2-tailed Pvalues are reported.

Throughout this document, various references are mentioned. All suchreferences are incorporated herein by reference, including thereferences set forth in the following list:

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It will be understood that various details of the presently disclosedsubject matter can be changed without departing from the scope of thesubject matter disclosed herein. Furthermore, the foregoing descriptionis for the purpose of illustration only, and not for the purpose oflimitation.

What is claimed is:
 1. A method of identifying an Alu RNA inhibitor,comprising: providing a cell in culture wherein Alu RNA is upregulated;contacting the cell with a candidate compound; and determining whetherthe candidate compound results in a change in the Alu RNA.
 2. The methodof claim 1, wherein the cell is an RPE cell.
 3. The method of claim 1,wherein the Alu RNA is upregulated by decreasing native levels of DICERpolypeptides in the cell; or by using heat shock stress.
 4. The methodof claim 1, wherein the change in the Alu RNA is a measurable decreasein Alu RNA, said change being an indication that the candidate compoundis an Alu RNA inhibitor.
 5. A method of protecting an RPE cell,comprising: inhibiting Alu RNA associated with the RPE cell.
 6. Themethod of claim 5, wherein the RPE cell is of a subject havingage-related macular degeneration.
 7. The method of claim 5, wherein theinhibiting Alu RNA comprises increasing levels of a DICER polypeptide inthe cell.
 8. The method of claim 7, where increasing levels of a DICERpolypeptide comprises overexpressing the DICER polypeptide in the cell.9. The method of claim 7, wherein increasing levels of a DICERpolypeptide comprises using a vector comprising a nucleotide encodingthe DICER polypeptide.
 10. The method of claim 9, wherein the vector isa viral vector or a plasmid vector.
 11. The method of claim 9, whereinthe nucleotide encoding the DICER polypeptide is selected from SEQ IDNO: 7 and SEQ ID NO:
 8. 12. The method of claim 7, wherein the DICERpolypeptide is selected from SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, 16,18, and
 20. 13. The method of claim 7, wherein the DICER polypeptidecomprises the sequence of SEQ ID NO: 9, 18, or 20, or a functionalfragment thereof.
 14. The method of claim 7, wherein the DICERpolypeptide comprises the following amino acid residues of thepolypeptide of SEQ ID NO: 9: 605-1922, 605-1912, 1666-1922, 1666-1912,605-1786 and 1800-1922, 605-1786 and 1800-1912, 1666-1786 and 1800-1922,1666-1786 and 1800-1912, 1276-1922, 1276-1912, 1276-1786 and 1800-1922,1276-1786, 800-1912, 1275-1824, or 1276-1824.
 15. The method of claim 7,wherein increasing levels of a DICER polypeptide comprises using DICERmRNA or a functional fragment thereof.
 16. The method of claim 15,wherein the DICER mRNA has the sequence of SEQ ID NO: 17, 19, or
 21. 17.The method of claim 15, wherein the DICER mRNA encodes a DICERpolypeptide selected from SEQ ID NO: 9, 18, or 20, or a functionalfragment thereof.
 18. The method of claim 5, wherein the inhibiting AluRNA comprises administering an oligonucleotide targeting Alu RNA. 19.The method of claim 18, wherein the oligonucleotide has the sequenceselected from SEQ ID NO: 22, 23, 24, 25, and
 26. 20. An isolatednucleotide molecule selected from: a double-stranded RNA molecule thatinhibits expression of Alu RNA, wherein a first strand of thedouble-stranded RNA comprises a sequence selected from SEQ ID NO: 1, 2,3, 4, 5, and 6 and including about 19 to 25 nucleotides; and anoligonucleotide that inhibits the expression of Alu RNA, comprising asequence selected from SEQ ID NO: 22, 23, 24, 25, and 26 and includingabout 29 to 100 nucleotides.